Huntington's Disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive impairments. The mutant huntingtin (htt) protein with expanded polyglutamine repeats are responsible for HD based on studies in human HD brains and in transgenic mice expressing mutant htt. Proteolytic fragments of htt are involved in the HD disease process. In human brain, N-terminal htt fragments in HD brains have been demonstrated. Expression of mutant N-terminal htt fragments in transgenic mice induces behavioral features and neuronal deficits that resemble HD. Notably, distinct patterns of htt N- and C-terminal fragments in human HD brains has been demonstrated. However, identification of these in vivo brain htt proteolytic fragments has not yet been determined. The critical, unsolved question is what are the primary sequences of htt proteolytic fragments in affected striatum and cortex of human HD brains? Therefore, the goal of this proposal will be to evaluate the primary sequences of N-terminal, N-domain, and C-domain htt fragments from human HD striatum and cortex, as well as from HD mouse models that express full-length htt. The first aim will evaluate the peptide sequences of in vivo htt fragments from mouse brain regions of transgenic HD mouse models that express full-length htt. Htt fragments will be purified by protein chromatographic resins that provide highly enriched htt proteins for purification;purification will include anti- htt affinity columns with well-characterized antisera that recognize the N-terminal region, N-domain, and C- domain areas of full-length htt. Purified htt fragments will be subjected to peptide sequencing by mass spectrometry, including sequencing of N- and C-terminal regions. Results will indicate peptide sequences of htt proteolytic fragments in mouse models of HD that express full-length htt. The second aim will evaluate the peptide sequences of human htt fragments purified from human HD brain regions - cortex and striatum - by mass spectrometry. The purification of htt fragments from human brain regions will utilize the purification procedure developed in aim 1 for mouse htt fragments. Mass spectrometry of purified human htt fragments will be conducted as described for htt fragments isolated from mouse brain. Results will provide key knowledge of the htt fragment sequences in human HD brain. Results of this project are essential for the next stages of HD research to define the most neurotoxic htt fragments, to elucidate the full spectrum of proteases that generate toxic htt fragments, and to move forward to drug discovery of protease inhibitors that may reduce production of htt fragments. This project will have extraordinarily high benefit for future development of effective therapeutic treatments for HD. PUBLIC HEALTH RELEVANCE: The goal of this project is to evaluate the primary peptide sequences of the in vivo mutant huntingtin (htt) protein fragments that are known to be responsible for the development of Huntington's disease (HD). These htt fragments in HD brains of patients have not yet been completely defined with respect to their primary amino acid sequences, which will be achieved in this project by their analyses by purification and current mass spectrometry approaches. The knowledge gained from this project is essential for understanding key htt mechanisms underlying HD, which will provide future strategies to improve the disease condition.